This proposal focuses on the development and evaluating of human anti-HIV- 1 antibodies. The antibodies prepared in these studies are intended for therapeutic and prophylactic application in HIV-1 infection. Their development and characterization will further our understanding of the molecular requirements for HIV-1 entry and vaccine design. Large panels of antibodies reactive with the envelope glycoproteins of primary HIV-1 isolates representative of each clade will be prepared from combinatorial antibodies, and to define and discover novel neutralizing epitopes such as the chemokine receptor binding site which is formed when env binds CD4. Human antibodies will be evolved from antibodies selected from animals which have been immunized with defined antigens and antigen combinations using recently developed methodologies. The breadth and potency of the anti-HIV-1 activity of the selected antibodies will be defined, as well as the epitope to which they bind. Selection of peptides which mimic neutralizing epitopes as well as elicit these antibodies will be studies as a vaccine approach. Structural studies of antibodies in complex with env and peptides will further define structural requirements for vaccine design. We will address the therapeutic and prophylactic potential of potent and broadly reactive human antibodies in a small animal model, the hu-PBL-SCID mouse. We will study immunotoxin conjugates of antibodies in hopes of developing a curative strategy when combined with existing multi- drug therapies. Cell lines which produce large amounts of these human antibodies will be constructed with the goal of making these practical pharmaceuticals. Cumulatively, these studies will provide a more thorough understanding HIV-1 envelope interactions, requirements for the induction of effective humoral responses in vaccine design, and will result in new molecules for the treatment and prevention of HIV-1 infection.